The present invention relates to novel peptide and nucleotide sequences. More particularly, the present invention relates to novel polypeptides capable of inhibiting at least in part the interaction between presenilin 1 or presenilin 2 on the one hand and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide on the other hand. The present invention also relates to the development of in vitro tests for the demonstration of molecules and in particular of small molecules capable of inhibiting this interaction.
The Axcex2 amyloid peptide, of 37 to 42 amino acids, is the principal protein component of characteristic senile plaques of Alzheimer""s disease. This peptide is produced by cleavage of its precursor, the precursor protein of amyloid peptide (APP). Mutations in the gene of the APP are responsible for certain early familial forms of Alzheimer""s disease. However, the majority of these forms are connected with the presence of mutations on two genes, presenilins PS1 (initially called S182) and PS2 (initially STM2), recently identified by positional cloning (Hardy, 1997). These forms are dominant and these anomalies all correspond to mis-sense mutations with the exception of one involving the deletion of an exon. The presenilins are hydrophobic membrane proteins of molecular mass of approximately 45-50 kDa and which have 67% identity between them. They are homologous with two proteins of C. elegans, SPE4 and Sel-12, which are respectively involved indirectly in intracellular transport and in the signalling of the Notch receptors. However, the physiological function of the presenilins is still unknown. Involvement in Alzheimer""s disease of two so closely related proteins suggests that the presenilins contribute to an essential physiological route in the aetiology of this pathology.
The protein PS1 comprises 467 amino acids and PS2 comprises 448. Both of them have the structure of a membrane protein with 6 to 8 potential transmembrane domains. Each of the presenilins is subject in vivo to precise proteolytic cleavage resulting in two fragments generally called N(amino)- and C(carboxy)-terminal fragments (Thinakaran et al., 1996). This cleavage has been mapped between the residues 291 and 299 of PS1 (Podlisny et al., 1997) and in a homologous region of PS2. N-terminal (N-ter) fragment is thus in general understood as meaning the fragment from position 1 to approximately 291 of PS1 and C-terminal fragment is understood as meaning the remainder. Although the exact topology of the presenilins in the lipid membranes has not been clearly established, it is proposed that their N- and C-terminal[RC1] ends, as well as the large hydrophilic loop, are present in the cytosol compartment (Doan et al., 1996, see Scheme FIG. 1).
It has now been demonstrated that the mutated forms of the presenilins induce the increase in the production of the long amyloid peptide Axcex2 1-42 with respect to that of Axcex2 1-40 as much in carrier patients (Scheuner et al., 1996), as in transfected cells (Borchelt et al., 1996) or in transgenic mice (Duff et al., 1996). The amyloid peptide Axcex2, which forms senile plaques, lesions characteristic of the pathology, and its different forms are derived from the catabolism of the amyloid precursor protein, APP. In particular, two essential forms of the amyloid peptide have been described, one of forty residues, Axcex240, and the other having two additional residues in its carboxy terminal, Axcex242. In vitro, the peptide Axcex2 has strong aggregation properties which are increased for the form Axcex242 and the latter effectively seems to form the first aggregates detectable in the pathology. In addition, the form Axcex242 is specifically produced after cranial trauma in man, which constitutes one of the best established environmental risk factors of Alzheimer""s disease. Moreover, the early genetic forms of the disease connected with mutations as much on APP (there are six of them) as now on the presenilins 1 and 2, all contribute to an increase in the Axcex242/Axcex240 ratio. All of these factors seem to point to Axcex242 as the key agent of the pathology as much in the genetic forms as in the sporadic forms of the disease and the elucidation of its mechanism of formation has become a fundamental question.
In this respect, complex formation in the same cell envelope between the precursor of the xcex2-amyloid peptide and PS1 or PS2 has been reported (Weidemann et al., 1997, Xia et al., 1997), however, the precise nature of the events responsible for the production of the xcex2-amyloid peptide is not known and no link has yet been able to be established between the possible role of these complexes and the production of the xcex2-amyloid peptide Axcex242. It is nevertheless important to note that the peptide Axcex242, but not Axcex240, seem to be localized in the endoplasmic reticulum in neuronal cells (Hartmann et al., 1997).
The present invention results from the identification and the characterization by the Applicant of particular regions of presenilin 1 (PS1) and of presenilin 2 (PS2), as well as of particular regions of the xcex2-amyloid precursor peptide (APP) involved in the formation of APP/PS1 and APP/PS2 complexes.
The present invention follows in particular from the demonstration of the capacity of the N-terminal hydrophilic region (amino acids 1-87) of PS2 to recognize different domains of APP. It additionally follows from the demonstration of similar properties for the N-terminal region of PS1 (fragment 1-213). It likewise results from the demonstration of the capacity of the polypeptides derived from the presenilin regions defined above to inhibit the formation of complexes between APP and the presenilins. The presenilins of the present application correspond essentially to presenilin 1 (PS1) and/or to presenilin 2 (PS2).
The present invention additionally results from the demonstration of the unexpected special cellular location of the regions in interaction with respect to the lipid membrane. It results more particularly from the fact that these interactions can take place not only at the membrane level but also at the level of the lumen of the endoplasmic reticulum and in the extracellular compartment. This is unexpected inasmuch as the N-terminal region of the PS (involved in the interaction) is generally considered as being localized in the cytoplasm under standard conditions.
The characterization of the domains of interaction of APP and of the presenilins and the demonstration of the different cellular locations of these interactions allow the preparation of novel polypeptides which can be utilized pharmaceutically to be envisaged.
A first subject of the invention thus relates to polypeptides capable of inhibiting at least in part the interaction between a presenilin and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide.
In the sense of capable of inhibiting the interaction, it is understood that the presence of the polypeptides of the invention and/or of the ligands and/or molecules demonstrated with the aid of the process of the invention suffice to inhibit at least partially the said interaction between a presenilin and/or its N-terminal end and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide and preferably the Axcex21-42 peptide.
It is demonstrated in the examples of the present application that the inhibition of this interaction with one of the polypeptides of the invention leads to a decrease in the production of the Axcex21-42 intracellular amyloid peptide. This functional consequence is thus envisaged for any polypeptide of the invention and/or ligands and/or molecules demonstrated with the aid of the process of the invention. To inhibit this interaction and thus to inhibit the production of Axcex21-42 as a consquence represents a therapeutic target of choice in the diseases involving this form of the amyloid peptide.
According to a particular embodiment, the polypeptides according to the invention have at least one part of presenilin 2 (PS2) allowing the interaction with the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide. Preferably, the polypeptides according to the invention are characterized in that the part of PS2 corresponds to the N-terminal hydrophilic fragment of PS2. More preferentially, the polypeptides according to the invention comprise all or part of the sequence corresponding to the sequence SEQ ID NO:2 or of a sequence derived from this.
According to another embodiment, the polypeptides according to the invention have at least one part of PS1 allowing interaction with the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide. Preferably, the polypeptides according to the invention comprise all or part of the sequence corresponding to the sequence SEQ ID NO:4 or of a sequence derived from this.
According to another embodiment, the polypeptides according to the invention comprise at least the common regions of homology corresponding to the sequences SEQ ID NO:2 and SEQ ID NO:4 respectively
According to another embodiment, the polypeptides according to the invention have at least one part of the precursor of the xcex2-amyloid peptide (APP). Preferably, the polypeptides according to the invention contain a part of APP outside the region corresponding to the xcex2-amyloid peptide. Even more preferably, the polypeptides are characterized in that the part of the precursor of the xcex2-amyloid peptide comprises all or part of the fragment 1-596. More preferably, the polypeptides according to the invention contain all or part of a sequence chosen from tamongst the sequence corresponding to the fragment 1-596 of the sequence SEQ ID NO:6, or a derived sequence.
In the sense of the present invention, the term derived polypeptide sequence denotes any polypeptide sequence differing from polypeptide sequences corresponding to the sequences presented in SEQ ID NO:2 or SEQ ID NO:4 respectively, or the denoted fragments of SEQ ID NO:6, obtained by one or more modifications of genetic and/or chemical nature, and having the capacity to inhibit at least in part the interaction between presenilin 1 or presenilin 2 and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide. Modification of genetic and/or chemical nature should be understood as meaning any mutation, substitution, deletion, addition and/or modification of one or more residues. Such derivatives can be generated with different aims, such as, especially, that of increasing the affinity of the peptide for its site of interaction, that of improving its level of production, that of increasing its resistance to proteases, that of increasing its therapeutic efficacy or of reducing its secondary effects, or that of conferring on it novel pharmacokinetic and/or biological properties.
The invention likewise provides non-peptide or not exclusively peptide compounds which can be used pharmaceutically. In fact, it is possible, starting from the polypeptide motifs described in the present application, to produce molecules which inhibit at least partially the interaction between presenilin 1 or presenilin 2 and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide, and which are not exclusively peptide and are compatible with pharmaceutical use. In this respect, the invention relates to the use of polypeptides such as described above for the preparation of non-peptide, or not exclusively peptide, pharmacologically active molecules by determination of the structural elements of these polypeptides which are important for their activity and reproduction of these elements by non-peptide or not exclusively peptide structures. The invention also relates to pharmaceutical compositions comprising one or more molecules prepared in this way.
The polypeptides according to the invention should comprise sequences allowing precise cellular localization in order to inhibit the interaction between the presenilins and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide. Preferentially, these are the polypeptides derived from SEQ ID NO:2 and SEQ ID NO:4 respectively which comprise exogenous cell localization sequences and more preferably still, a polypeptide comprising the N-terminal end of PS1 or PS2. Amongst these sequences, it is possible to mention signal peptide sequences such as the signal peptide sequence of IgkB, the signal peptide of APP, the signal peptides of subunits of the nicotinic receptors of muscular and central acetylcholine etc. . . .
Among the polypeptides of particular interest, there may be mentioned a polypeptide comprising the 87 first residues of the N-terminal end of PS2 and the IgkB signal peptide.
The present invention likewise relates to any nucleotide sequence coding for a peptide capable of inhibiting at least in part the interaction between presenilin 1 or presenilin 2 and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide.
According to a particular embodiment, it is a nucleotide sequence comprising all or part of the nucleotide sequence SEQ ID NO:1 or of a sequence derived from this. According to another embodiment, it is a nucleotide sequence comprising all or part of the nucleotide sequence SEQ ID NO:3 or of a sequence derived from this. Preferably, it is a nucleotide sequence comprising the zones of homologies common to the nucleotide sequences SEQ ID NO:1 and SEQ ID NO:3 respectively According to another embodiment, it is the nucleotide sequence corresponding to the fragment 1-596 (nucleic acids 1 to 1788) of the sequence SEQ ID NO:6(SEQ ID NO:5), or a derived sequence.
In the sense of the present invention, the term derived nucleotide sequence denotes any sequence different from the considered sequence because of the degeneration of the genetic code, obtained by one or more modifications of genetic and/or chemical nature, as well as any sequence hybridizing with these sequences or fragments of these and coding for a polypeptide according to the invention. Modification of genetic and/or chemical nature can be understood as meaning any mutation, substitution, deletion, addition and/or modification of one or more residues. The term derivative likewise comprises the sequences homologous with the considered sequence, originating from other cell sources and especially from cells of human origin, or from other organisms. Such homologous sequences can be obtained by hybridization experiments. The hybridizations can be carried out starting from nucleic acid banks, using, as a probe, the native sequence or a fragment of this, under variable hybridization conditions (Maniatis et al., 1982).
Nucleotide sequences according to the invention can be of artificial or non-artificial origin. They can be genomic sequences, of cDNA, of RNA, of hybrid sequences or of synthetic or semi-synthetic sequences. These sequences can be obtained, for example, by screening DNA banks (cDNA bank, genomic DNA bank) by means of probes elaborated on the basis of sequences shown above. Such banks can be prepared starting from cells of different origins by conventional techniques of molecular biology known to the person skilled in the art. The nucleotide sequences of the invention can likewise be prepared by chemical synthesis or even by mixed methods including the chemical or enzymatic modification of sequences obtained by screening of banks. Generally speaking, the nucleic acids of the invention can be prepared according to any technique known to the person skilled in the art.
Another subject of the present invention relates to a process for the preparation of the polypeptides of the invention according to which a cell containing a nucleotide sequence according to the invention is cultured under conditions of expression of the said sequence and the polypeptide produced is recovered. In this case, the part coding for the said polypeptide is generally placed under the control of signals allowing its expression in a cell host. The choice of these signals (promoters, terminators, secretion leader sequence, etc.) can vary as a function of the cell host used. In addition, the nucleotide sequences of the invention can be part of a vector which can be an autonomous or integrative replication vector. More particularly, autonomous replication vectors can be prepared using autonomous replication sequences in the chosen host. When integrative vectors are involved, these can be prepared, for example, using sequences homologous to certain regions of the genome of the host, allowing, by homologous recombination, the integration of the vector.
The present invention likewise relates to host cells transformed with a nucleic acid having a nucleotide sequence according to the invention. The cell hosts which can be used for the production of the peptides of the invention by the recombinant route are eukaryotic or prokaryotic hosts as well. Amongst the eukaryotic hosts which are suitable, it is possible to mention animal cells, yeasts or fungi. In particular, when yeasts are concerned, it is possible to mention yeasts of the genus Saccharomyces, Kluyveromyces, Pichia, Schwanniomyces or Hansenula. When they are animal cells, it is possible to mention COS, CHO, C127, human neuroblastoma cells etc. Amongst the fungi, it is more particularly possible to mention Aspergillus ssp. or Trichoderma ssp. As prokaryotic hosts, it is preferred to use the following bacteria: E. coli, Bacillus or Streptomyces.
According to a preferred embodiment, the host cells are advantageously represented by recombinant yeast strains for the expression of the nucleic acids of the invention as well as the production of the proteins derived from these.
Preferentially, the host cells comprise at least one sequence or a sequence fragment chosen from amongst the sequences SEQ ID NO:1 or SEQ ID NO:3 respectively, or the denoted fragments of SEQ ID NO:5 for the production of polypeptides according to the invention.
The nucleotide sequences according to the invention can be used in the context of gene therapy, in particular through the addition of a signal peptide for the derivatives of SEQ ID NO:1 and SEQ ID NO:3 respectively, for the production and transfer in vivo of polypeptides capable of inhibiting at least in part the interaction between presenilin 1 or presenilin 2 and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide. In fact, unexpectedly, it has been demonstrated in the present application that a signal peptide is necessary for the addressing of the polypeptides of the invention in the lumen of the endoplasmic reticulum and thus for conferring on the polypeptides derived from the sequences SEQ ID NO:2 and SEQ ID NO:4 respectively a biological activity with the aim of inhibiting the interaction between the presenilins and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide.
According to another embodiment of the invention, the nucleotide sequences of the invention are used for the construction of an expression cassette, which can be used in an expression vector. In particular, the expression cassette serves for the production of the polypeptides according to the invention.
The polypeptides of the invention can be obtained by expression in a cell host of a nucleotide sequence such as described above, incorporated or not incorporated in a recombinant DNA, using techniques known to the person skilled in the art, or by a combination of these techniques.
Preferentially, the nucleic sequences according to the invention are part of a vector which is useful for inducing in vivo, ex vivo and/or in vitro the expression of the claimed polypeptides. The vector used can be of various origins, since it is capable of transforming animal cells, preferably human nerve cells. It can be a viral vector, non-viral vector or a plasmid vector. In a preferred embodiment of the invention, a viral vector is used which can be derived from adenoviruses, retroviruses, adeno-associated viruses (AAVs), from herpesvirus, from cytomegalovirus (CMV), from vaccinia virus, etc. Vectors derived from adenoviruses, from retroviruses or from AAVs incorporating heterologous nucleic acid sequences have been described in the literature [Akli et al., Nature Genetics 3 (1993) 224; Stratford-Perricaudet et al., Human Gene Therapy 1 (1990) 241; EP 185 573, Levrero et al., Gene 101 (1991) 195; Le Gal la Salle et al., Science 259 (1993) 988; Roemer and Friedmann, Eur. J. Biochem. 208 (1992) 211; Dobson et al., Neuron 5 (1990) 353; Chiocca et al., New Biol. 2 (1990) 739; Miyanohara et al., New Biol. 4 (1992) 238; WO91/18088].
The present invention thus likewise relates to any recombinant virus comprising, inserted in its genome, a nucleic sequence such as defined above coding for a polypeptide of the invention.
Advantageously, the recombinant virus according to the invention is a defective virus. The term xe2x80x9cdefective virusxe2x80x9d denotes a virus incapable of replicating in the target cell. Generally, the genome of the defective viruses used within the context of the present invention is thus devoid at least of the sequences necessary for the replication of the said virus in the infected cell. These regions can be either eliminated (all or in part), rendered non-functional, or substituted by other sequences and especially by the nucleic acid of the invention.
Preferentially, the defective virus nevertheless conserves the sequences of its genome which are necessary for the encapsidation of the viral particles.
It is particularly advantageous to use the nucleic sequences of the invention in incorporated form with an adenovirus, an AAV or a defective recombinant retrovirus. According to a preferred embodiment, it is an adenovirus.
Different serotypes of adenovirus exist whose structure and properties vary somewhat. Amongst these serotypes, it is preferred to use within the context of the present invention the human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or the adenoviruses of animal origin (see Application WO94/26914). Amongst the adenoviruses of animal origin which can be used within the context of the present invention, it is possible to mention the adenoviruses of canine, bovine, murine, (example: Mav1, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or even simian origin (example: SAV). Preferably, the adenovirus of animal origin is a canine adenovirus, more preferentially a CAV2 adenovirus [manhattan or A26/61 strain (ATCC VR-800) for example]. Preferably, adenoviruses of human or canine or mixed origin are used within the context of the invention. Preferentially, in the genome of the adenoviruses of the invention, the region E1 at least is non-functional. The viral gene considered can be rendered non-functional by any technique known to the person skilled in the art, and especially by total suppression, substitution, partial deletion, or addition of one or more bases in the gene or genes considered. Other regions can likewise be modified, and especially the E3 (WO95/02697), E2 (WO94/28938), E4 (WO94/28152, WO94/12649, WO95/02697) and L5 (WO95/02697) region. According to a preferred embodiment, the adenovirus comprises a deletion in the regions E1 and E4. According to another preferred embodiment, it comprises a deletion in the region E1 at the level of which are inserted the region E4 and the coding sequence. In the viruses of the invention, the deletion in the region E1 preferentially extends from nucleotides 455 to 3329 in the sequence of the adenovirus Ad5. According to another preferred embodiment, the exogenous nucleic acid sequence is inserted at the level of the deletion in the E1 region.
The defective recombinant viruses of the invention can be prepared by homologous recombination between a defective virus and a plasmid carrying, inter alia, the nucleotide sequence such as defined above (Levrero et al., Gene 101 (1991) 195; Graham, EMBO J. 3(12) (1984) 2917). Homologous recombination takes place after cotransfection of the said viruses and plasmid in an appropriate cell line. The cell line used must preferably (i) be transformable by the said elements, and (ii) contain sequences capable of complementing the part of the genome of the defective virus, preferably in integrated form to avoid the risks of recombination. By way of example of a line which can be used for the preparation of defective recombinant adenoviruses, it is possible to mention the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which especially contains, integrated in its genome, the left part of the genome of an Ad5 adenovirus (12%). By way of example of a line which can be used for the preparation of defective recombinant retroviruses, it is possible to mention the CRIP line (Danos and Mulligan, PNAS 85 (1988) 6460). Next, the viruses which have multiplied are recovered and purified according to the conventional techniques of molecular biology.
The present application likewise relates to defective recombinant viruses comprising a heterologous nucleic sequence coding for a polypeptide according to the invention.
Another subject of the invention resides in polyclonal or monoclonal antibodies or antibody fragment. Such antibodies can be generated by methods known to the person skilled in the art. In particular, these antibodies can be prepared by immunization of an animal against a polypeptide whose sequence is chosen from amongst the sequences SEQ ID NO:2 or SEQ ID NO:4 respectively, or the denoted fragments of SEQ ID NO:6, then taking of a sample of the blood and isolation of the antibodies. These antibodies can likewise be generated by preparation of hybridomas according to techniques known to the person skilled in the art. The antibodies or antibody fragment according to the invention can especially be used to inhibit at least in part the interaction between presenilin 1 or presenilin 2 and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide.
Another object of the present invention relates to a process for identifying compounds capable of modulating or of inhibiting at least in part the interaction between presenilin 1 or presenilin 2 and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide. In particular, the process can be used as a test for screening molecules to identify such inhibitor compounds.
This test is based, in particular, on the detection of the inhibition of the interaction between the presenilins (1 or 2) and the APP or the Axcex2 peptide generally and between the Axcex21-42 peptide and the N-terminal end of PS2 particularly. In fact, with the aid of marker proteins attached to the presenilins or fragments of these and appropriate visualization systems and especially by immunoprecipitation, use of chromophores or fluophores, it is quite possible to detect an inhibition in the interaction of the proteins or fragments of these mentioned above. Such a process thus comprises at least one labelling step of the presenilins and/or of the APP or of the fragments of these and a step of detection of the inhibition of the interaction either between the Axcex21-42 peptide and the N-terminal end of the presenilins and preferentially PS2, or between the complete APP proteins and presenilins.
According to a first embodiment of the process, the demonstration and/or identification of such compounds is carried out according to the following steps:
The Axcex21-42 peptide is absorbed beforehand on a nitrocellulose membrane by incubation.
a bacterial extract containing all or part of a presenilin (PS1 or PS2) and advantageously the N-terminal end, is then added for incubation with the molecule or a mixture containing different molecules to be tested
After washing, the interaction of the presenilin with the Axcex21-42 peptide on the nitrocellulose filter is demonstrated with the aid of presenilin marker proteins. The molecules sought inhibit the interaction and thus decrease the intensity of the signal of the marker proteins.
The marker proteins used are advantageously
a) the immobilizing protein of the S-tag, coupled to alkaline phosphatase or to a fluorescent chromophore, or
b) an anti-PSNT antibody, that is to say directed against the N-terminal end of a presenilin.
According to another embodiment of the process, the investigation of novel compounds is carried out in the following manner:
the Axcex242 peptide is incubated beforehand on a plate containing wells (96-well format or greater)
the N-terminal end of a purified recombinant presenilin is then added with the molecule or a mixture containing different molecules to be tested, for incubation
After washing, the interaction of presenilin with the Axcex21-42 peptide in the plate is demonstrated with the aid of presenilin marker proteins. The loss of interaction between the presenilins and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide is detected by spectrophotometry.
In the precise case of the use of the immobilizing protein of the S-tag coupled to alkaline phosphatase as marker protein, after visualization with a colorimetric substrate, the signal is detected at 450 nm.
According to an advantageous and preferred embodiment of the process, the demonstration and/or the isolation of compounds capable of modulating or of inhibiting at least in part the interaction generally between the presenilin 1 or the presenilin 2 and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide particularly between the Axcex21-42 peptide and the N-terminal end of PS2 is carried out according to the following steps:
a molecule or a mixture containing different molecules is contacted with the Axcex21-42 peptide synthesized with a biotin and an arm of 3 xcex2-alanines (or of 3 lysines) at its N-terminal end (upstream of position 1)
the above reaction mixture is incubated with the N-terminal end of a purified presenilin labelled with the aid of a first fluorophore. Advantageously, the fluophore is europium cryptate
streptavidin (which will be immobilized on the biotin of the biot-Axcex21-42 peptide) coupled to a second fluophore capable of being excited at the emission wavelength of the first fluophore is added such that it benefits from a fluorescence transfer if the two fluorophores are found in close proximity.
the demonstration of the novel compounds inhibiting the interaction is detected by fluorometry at the emission wavelength of the first fluophore and/or by measuring the decrease in the signal at the emission wavelength of the second fluophore
According to a particular embodiment, this second fluophore is XL665 which is allophycocyanin crosslinked chemically to increase its fluorescence at 665 nm (CisBiointernational). The loss of interaction is thus detected by fluorometery at the emission wavelength of the first fluophore and by the decrease in the signal of XL665 whose emission wavelength is at 665 nm.
This process is quite advantageous because it allows the interaction between the presenilins and the APP and/or the Axcex2 peptide in liquid and homogeneous phase to be demonstrated directly and consequently the inhibitory molecules of the said interaction to be demonstrated. In fact, this process is based on the transfer of fluorescence between two fluophores if these two chromophores are in physical proximity (thus in the case of interaction between Axcex21-42 and the recombinant protein). According to a preferred variant of the process, the first fluorophore is europium cryptate, carried by the labelled recombinant protein (excited at 337 nm) reacting with the streptavidin-XL665 immobilized on the biot-Axcex2 peptide, and in particular the biot-Axcex21-42 peptide. Advantageously, the labelled protein is formed by the N-terminal end of one or the other of the presenilins (PSNT-K). The loss of the fluorescence at 665 nm and the increase in the fluorescence at 620 nm characteristic of the europium cryptate indicates an inhibition of the interaction between the presenilins or their N-terminal ends and the APP and/or the Axcex2 peptide by the molecules sought.
According to a variant of this process, the molecule or the mixture containing the different molecules can be first contacted with the N-terminal end of a labelled purified presenilin with the aid of europium cryptate (PSNT-K) and then with the Axcex21-40 or Axcex21-42 peptide carrying a biotin and an arm of 3 xcex2-alanines (or of 3 lysines) at their N-terminal end. The demonstration of novel molecules capable of modulating or of inhibiting at least in part the interaction between the presenilin 1 or the presenilin 2 and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide will be likewise made, after addition of the streptavidin labelled on the XL665, by spectrofluorometry according to the method above and in particular by reading of the fluorescence at 665 nm.
According to a last embodiment of the process of demonstrating compounds inhibiting the interaction generally between the presenilins (1 or 2) and the APP or the Axcex2 peptide and particularly between the Axcex21-42 peptide and the N-terminal end of PS2 has the following steps:
a mixture a) of cell lysates containing all or part of a presenilin (PS1 or PS2) and advantageously the N-terminal end, b) of cell lysates containing the APP, lysates obtained starting from cells infected by viruses and in particular by baculoviruses and c) the molecule or a mixture containing different molecules to be tested are contacted
the proteins solubilized and corresponding to the presenilins or to the APP or the A xcex2peptide are co-immunoprecipitated with the aid of antibodies which are appropriate and well-known to the person skilled in the art
the loss of the co-immunoprecipitation of the presenilins and of the APP is visualized by Western blot with marker antibodies indicating that the molecules tested have the sought inhibitory property
In a particular embodiment, the processes of the invention described above are adapted to the demonstration and/or the isolation of ligands, agonists or antagonists of the interaction between the presenilins and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide.
The present invention likewise relates to the use of the polypeptides defined above for the demonstration of ligands of the polypeptides but above all of ligands of the presenilins, of the precursor of the xcex2-amyloid peptide and/or of the xcex2-amyloid peptide, and preferentially of the Axcex21-42 peptide and/or of the N-terminal end of PS2, as well as of compounds capable of inhibiting at least in part the interaction between a presenilin and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide.
Another subject of the invention relates to the use of a ligand or of a modulator identified and/or obtained according to the processes described above as medicament. Such ligands or modulators by means of their capacity to interfere at the level of the interaction between the presenilins and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide can thus modulate the production of the amyloid peptide Axcex21-42 and allow certain neurological disorders and especially Alzheimer""s disease to be treated.
Another subject of the invention relates to the perfection of an interaction test between a presenilin and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide, and preferentially between the Axcex21-42 peptide and the N-terminal end of PS2, characterized in that it comprises at least one fluorescence transfer step between two fluophores immobilized on the preceding molecules and a step of visualization of the interaction measured by spectrofluorometry. As mentioned above, this test is also used to demonstrate molecules which inhibit the said interaction, according to the method for detecting the inhibition of the interaction, described in the present application.
The invention additionally relates to any pharmaceutical composition comprising as active principle at least one polypeptide such as defined above.
It also relates to any pharmaceutical composition comprising as active principle at least one antibody or antibody fragment such as defined above, and/or an antisense oligonucleotide and/or a ligand such as defined above. The invention likewise relates to any pharmaceutical composition comprising as active principle at least one nucleotide sequence such as defined above.
In addition, it also relates to the pharmaceutical compositions in which the peptides, antibodies, ligands and nucleotide sequences defined above are associated with each other or with other active principles.
It likewise relates to the.compositions in which the nucleotide sequences according to the invention are incorporated in a recombinant viral or non-viral vector.
The pharmaceutical compositions according to the invention can be used to inhibit at least in part the interaction between a presenilin and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide. They are more preferentially pharmaceutical compositions intended for the treatment of neurodegenerative diseases such as, for example, Alzheimer""s disease.
Another subject of the present invention is the use of the polypeptides described previously to inhibit at least in part the interaction between a presenilin and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide and preferably the use of these polypeptides to obtain a medicament intended for the treatment of neurodegenerative diseases and especially of Alzheimer""s disease.
For their use according to the present invention, the polypeptides of the invention on the one hand or any molecule capable of inhibiting at least in part the interaction between a presenilin and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide, the corresponding nucleic sequences on the other hand or additionally the vectors such as described above are preferentially associated with one or more pharmaceutically acceptable vehicles to be formulated with a view to administration by the topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular or transdermal route, etc. Preferably, they are used in an oral form. The injectable form can nevertheless be envisaged and could in particular be formulated with sterile, isotonic saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc, or mixtures of such salts), or dry, especially lyophilized, compositions which, according to the case, allow the formation of injectable solutions by addition of sterilized water or of physiological serum.
The doses of vector and in particular of a virus used for the administration can be adapted as a function of different parameters, and especially as a function of the site of administration considered (organ, nervous or muscular tissue), of the number of injections, of the gene to be expressed, or even of the duration of the treatment sought. Generally, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 104 and 1014 pfu, and preferably 106 to 1010 pfu.
The term pfu (xe2x80x9cplaque forming unitxe2x80x9d) corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 15 days, the number of plaques of infected cells. The techniques for the determination of the pfu titre of a viral solution are well documented in the literature.
The present invention offers an efficacious means for treating the diseases in which the interaction between a presenilin and the precursor of the xcex2-amyloid peptide and/or the xcex2-amyloid peptide is involved and preferably for the treatment of neurodegenerative diseases and especially of Alzheimer""s disease.
The present invention will be more fully detailed with the aid of the examples below, which are considered as being of descriptive and non-limiting manner.